Human RECQL5 helicase; A Target Enabling Package

<p>RECQL5 is a member of the RecQ family of helicase which have important functions in DNA repair pathways and maintenance of genome integrity. RECQL5 has recently been identified as a synthetic lethal candidate in various haematological malignancies and has been verified by knockdown to sensitize myeloproliferative neoplasms (MPN) to DNA damaging agents. In this TEP we have expressed purified and determined the first ever crystal structures of RECQL5, in both APO and ADP/Mg2+ bound forms which crystallize in two distinctly different conformations. In vitro DNA stimulated ATPase assays suitable for high throughput screening have been developed as well as lower throughput orthogonal assays to verify potential hits. A fragment screening campaign has been initiated and single fragment hit has identified a potential allosteric site that may be targeted to block the transition between conformations that it thought to be part of the helicase mechanism. Finally included as part of the package we present 3 validated RECQL5 binding nanobodies, one of which is a potent inhibitor of RECQL5 ATPase activity and is suitable for use as a tool reagent to investigate inhibition of RECQL5 and its complexes in vitro.</p

Human RECQL5 helicase; A Target Enabling Package

<p>RECQL5 is a member of the RecQ family of helicase which have important functions in DNA repair pathways and maintenance of genome integrity. RECQL5 has recently been identified as a synthetic lethal candidate in various haematological malignancies and has been verified by knockdown to sensitize myeloproliferative neoplasms (MPN) to DNA damaging agents. In this TEP we have expressed purified and determined the first ever crystal structures of RECQL5, in both APO and ADP/Mg2+ bound forms which crystallize in two distinctly different conformations. In vitro DNA stimulated ATPase assays suitable for high throughput screening have been developed as well as lower throughput orthogonal assays to verify potential hits. A fragment screening campaign has been initiated and single fragment hit has identified a potential allosteric site that may be targeted to block the transition between conformations that it thought to be part of the helicase mechanism. Finally included as part of the package we present 3 validated RECQL5 binding nanobodies, one of which is a potent inhibitor of RECQL5 ATPase activity and is suitable for use as a tool reagent to investigate inhibition of RECQL5 and its complexes in vitro.</p

Human RECQL5 helicase; A Target Enabling Package

<p>RECQL5 is a member of the RecQ family of helicase which have important functions in DNA repair pathways and maintenance of genome integrity. RECQL5 has recently been identified as a synthetic lethal candidate in various haematological malignancies and has been verified by knockdown to sensitize myeloproliferative neoplasms (MPN) to DNA damaging agents. In this TEP we have expressed purified and determined the first ever crystal structures of RECQL5, in both APO and ADP/Mg2+ bound forms which crystallize in two distinctly different conformations. In vitro DNA stimulated ATPase assays suitable for high throughput screening have been developed as well as lower throughput orthogonal assays to verify potential hits. A fragment screening campaign has been initiated and single fragment hit has identified a potential allosteric site that may be targeted to block the transition between conformations that it thought to be part of the helicase mechanism. Finally included as part of the package we present 3 validated RECQL5 binding nanobodies, one of which is a potent inhibitor of RECQL5 ATPase activity and is suitable for use as a tool reagent to investigate inhibition of RECQL5 and its complexes in vitro.</p

Type II Inhibitors Targeting CDK2

Kinases can switch between active and inactive conformations of the ATP/Mg²⁺ binding motif DFG, which has been explored for the development of type I or type II inhibitors. However, factors modulating DFG conformations remain poorly understood. We chose CDK2 as a model system to study the DFG in–out transition on a target that was thought to have an inaccessible DFG-out conformation. We used site-directed mutagenesis of key residues identified in structural comparisons in conjunction with biochemical and biophysical characterization of the generated mutants. As a result, we identified key residues that facilitate the DFG-out movement, facilitating binding of type II inhibitors. However, surprisingly, we also found that wild type CDK2 is able to bind type II inhibitors. Using protein crystallography structural analysis of the CDK2 complex with an aminopyrimidine-phenyl urea inhibitor (K03861) revealed a canonical type II binding mode and the first available type II inhibitor CDK2 cocrystal structure. We found that the identified type II inhibitors compete with binding of activating cyclins. In addition, analysis of the binding kinetics of the identified inhibitors revealed slow off-rates. The study highlights the importance of residues that may be distant to the ATP binding pocket in modulating the energetics of the DFG-out transition and hence inhibitor binding. The presented data also provide the foundation for a new class of slow off-rate cyclin-competitive CDK2 inhibitors targeting the inactive DFG-out state of this important kinase target

Type II Inhibitors Targeting CDK2

Kinases can switch between active and inactive conformations of the ATP/Mg²⁺ binding motif DFG, which has been explored for the development of type I or type II inhibitors. However, factors modulating DFG conformations remain poorly understood. We chose CDK2 as a model system to study the DFG in–out transition on a target that was thought to have an inaccessible DFG-out conformation. We used site-directed mutagenesis of key residues identified in structural comparisons in conjunction with biochemical and biophysical characterization of the generated mutants. As a result, we identified key residues that facilitate the DFG-out movement, facilitating binding of type II inhibitors. However, surprisingly, we also found that wild type CDK2 is able to bind type II inhibitors. Using protein crystallography structural analysis of the CDK2 complex with an aminopyrimidine-phenyl urea inhibitor (K03861) revealed a canonical type II binding mode and the first available type II inhibitor CDK2 cocrystal structure. We found that the identified type II inhibitors compete with binding of activating cyclins. In addition, analysis of the binding kinetics of the identified inhibitors revealed slow off-rates. The study highlights the importance of residues that may be distant to the ATP binding pocket in modulating the energetics of the DFG-out transition and hence inhibitor binding. The presented data also provide the foundation for a new class of slow off-rate cyclin-competitive CDK2 inhibitors targeting the inactive DFG-out state of this important kinase target

Human Lysine Demethylase JMJD2D (KDM4D); A Target Enabling Package

<p>There are 4 members of the Lysine Demethylase JMJD2 (KDM4) family. SGC Oxford has expressed, purified and crystallized the catalytic domains of JMJD2A, JMJD2B, JMJD2C and JMJD2D as part of the probe programme. Fragment screening and X-ray crystallography identified a large number of binders, some of which were progressed into a medicinal chemistry programme. Despite significant effort molecules with probe properties were not obtained. Consequently it has been decided to put the information generated into the public domain.</p

Human Lysine Demethylase JMJD1B (KDM3B); A Target Enabling Package

<p>There are 3 members of the Lysine Demethylase JMJD1 (KDM3) family, JMJD1A-C. SGC Oxford has expressed, purified and crystallized the catalytic domains of JMJD1A, JMJD1B and JMJD1C as part of the probe programme. Fragment screening and X-ray crystallography identified a large number of binders, some of which were progressed into a medicinal chemistry programme. Despite significant effort molecules with probe properties were not obtained. Consequently it has been decided to put the information generated into the public domain.</p

Human Lysine Demethylase JMJD2D (KDM4D); A Target Enabling Package

<p>There are 4 members of the Lysine Demethylase JMJD2 (KDM4) family. SGC Oxford has expressed, purified and crystallized the catalytic domains of JMJD2A, JMJD2B, JMJD2C and JMJD2D as part of the probe programme. Fragment screening and X-ray crystallography identified a large number of binders, some of which were progressed into a medicinal chemistry programme. Despite significant effort molecules with probe properties were not obtained. Consequently it has been decided to put the information generated into the public domain.</p

Discovery of a Chemical Tool Inhibitor Targeting the Bromodomains of TRIM24 and BRPF

TRIM24 is a transcriptional regulator as well as an E3 ubiquitin ligase. It is overexpressed in diverse tumors, and high expression levels have been linked to poor prognosis in breast cancer patients. TRIM24 contains a PHD/bromodomain offering the opportunity to develop protein interaction inhibitors that target this protein interaction module. Here we identified potent acetyl-lysine mimetic benzimidazolones TRIM24 bromodomain inhibitors. The best compound of this series is a selective BRPF1B/TRIM24 dual inhibitor that bound with a <i>K</i>_D of 137 and 222 nM, respectively, but exerted good selectivity over other bromodomains. Cellular activity of the inhibitor was demonstrated using FRAP assays as well as cell viability data

Design of a Chemical Probe for the Bromodomain and Plant Homeodomain Finger-Containing (BRPF) Family of Proteins

The bromodomain and plant homeodomain finger-containing (BRPF) family are scaffolding proteins important for the recruitment of histone acetyltransferases of the MYST family to chromatin. Here, we describe <b>NI-57</b> (<b>16</b>) as new pan-BRPF chemical probe of the bromodomain (BRD) of the BRPFs. Inhibitor <b>16</b> preferentially bound the BRD of BRPF1 and BRPF2 over BRPF3, whereas binding to BRD9 was weaker. Compound <b>16</b> has excellent selectivity over nonclass IV BRD proteins. Target engagement of BRPF1B and BRPF2 with <b>16</b> was demonstrated in nanoBRET and FRAP assays. The binding of <b>16</b> to BRPF1B was rationalized through an X-ray cocrystal structure determination, which showed a flipped binding orientation when compared to previous structures. We report studies that show <b>16</b> has functional activity in cellular assays by modulation of the phenotype at low micromolar concentrations in both cancer and inflammatory models. Pharmacokinetic data for <b>16</b> was generated in mouse with single dose administration showing favorable oral bioavailabilit